Adult stem cells comprise a reservoir of cells with a remarkable capacity to proliferate and repair tissue damage. Hematopoietic stem cells (HSCs) are capable of extensive self-renewal in vivo and are successfully employed clinically to treat hematopoietic malignancies, yet in culture this self-renewal capacity is lost. While HSCs are one of the most extensively studied adult stem cells, many of the mechanisms that control their behavior remain to be elucidated. A major challenge facing stem cell biologists is an understanding of the mechanisms that direct the delicate balance between quiescence, self-renewal, and differentiation of HSCs both in vivo and in culture. The microenvironment, or niche, surrounding the stem cell is presumed to regulate its maintenance and self-renewal in vivo. It follows that HSCs plated in culture begin to specialize, likely due to loss of interaction with their native microenvironment. Accordingly, Specific Aim 1 is to study the influence of extrinsic factors on the maintenance of HSC function in culture. Bioengineered artificial microenvironments consisting of arrays of hydrogel microwells will be employed to enable exposure of large replicates of single cells to: (a) soluble proteins, (b) extracellular matrix proteins, and (c) ectodomains of transmembrane proteins. A novel protein tethering technology has been developed that facilitates physiological presentation of both extracellular matrix and ectodomain proteins through tethering to the bottom of each microwell; this advance enables studies of both ECM and cell-cell interactions characteristic of adult stem cell niches without the complexity of coculture. In vivo assays will be used to test the hypothesis that repopulation potential of cultured cells can be correlated with in vitro proliferation behavior. Therefore, Specific Aim 2 is to investigate the mechanisms of action of candidate extrinsic factors on HSC self-renewal. The following potential self-renewal mechanisms will be investigated: asymmetric divisions, symmetric divisions and reversion of transiently amplifying progenitors resulting in reacquisition of stem cell function. These studies will examine behavior and morphology at a single cell level to establish parameters that correlate with each self-renewal mechanism. Finally, the goal of Specific Aim 3 is to identify in vitro predictive parameters that correlate with HSC expansion and to use these parameters to screen for novel proteins. The immense statistical power of the microwell platform will be used to assay a large number of replicate clones to establish a subset of predictive parameters capable of identifying in vitro HSC expansion. The identified parameters will be used to identify novel ectodomain proteins capable of supporting expansion of HSCs in culture precluding the need for expensive in vivo assays. The ultimate goal is to increase both our understanding of HSC biology and the clinical utility of HSCs as a blood source.